This project is designed to define the morphologic, molecular, and metabolic characteristics of breast ducts and ductal epithelial cells at normal risk and increased risk for breast cancer. This information is needed to define early changes in the carcinogenic pathway for breast cancer, to develop an improved classification and molecular signature of preneoplastic breast tissue for risk assessment, and to identify new targets, and facilitate selection and monitoring of women, for breast cancer prevention. This project is developed by the following clinical and laboratory studies: a.) Protocol 02-C-0077, Characterization of High Risk Breast Duct Epithelium by Cytology, Breast Duct Endoscopy, and Gene Expression Profile (DN Danforth, PI). b.) Protocol 02-C-0144, Establishment of Normal Breast Epithelial Cell Cultures, and a High Risk Cell Line and Tissue Repository from Breast Tissue from Women at High Risk for Breast Cancer (DN Danforth, PI). c.) Comprehensive literature review of molecular changes in normal breast tissue at normal risk or at high risk for breast cancer, in hyperplasia, and in atypical hyperplasia. d.) Regulation of proliferation and DNA damage repair in breast epithelium by endogenous risk factors. Protocol 02-C-0077 examines and characterizes by ductal lavage and ductal endoscopy the ducts and ductal epithelium of women at normal risk and increased risk for breast cancer. Fifty-four women have been studied, 39 high risk subjects and 15 at subjects at normal risk. Cytologic atypia of ductal epithelial cells was identified and correlated with ductal endoscopic findings of architectural changes. Repeat ductal lavage and endoscopy was performed on all subjects with atypia to define the persistence of cellular and ductal changes. A new method for acquisition of ductal epithelial cellular samples for cytologic and molecular analysis was developed: ductal endoscopic sampling with brush sampling devices of normal epithelium. This provided significantly increased cellular yields and samples of more than 90% pure ductal epithelial cells. Molecular characteristics of high risk breast epithelium is in progress, and a novel method of whole genome amplification of DNA from cytopathologic slides has been developed. Initial studies will define abnormalities of the p53 gene, a major factor in breast carcinogenesis. P53 mutations will be defined by whole gene sequencing, segmental chromosomal loss in the 17p13.1 region by fine tiling CGH of chromosome 17, and allelic imbalance and intragenic loss of heterozygosity by SNP Taqman genotyping assay. Molecular analysis will then be expanded to include DNA methylation array to define tumor suppressor gene loss, an early change in breast tumorigenesis. (Danforth DN et al. Jour Surg Oncol, 94:555-564, 2006). Protocol, 02-C-0144 (DN Danforth, PI) establishes a tissue and cell line repository from all major sites of normal breast tissue at increased risk for breast cancer, including the contralateral normal breast, tissue adjacent to a breast cancer, women with a strong family history of breast cancer (including BRCA1 and BRCA2 mutation carriers), a Gail model risk estimate of breast cancer of more than 1.67%, or women with prior mediastinal irradiation for lymphoma. Tissues are processed to allow for a wide spectrum of molecular studies. Mortal epithelial, fibroblast and adipose cell lines are developed to allow for a wide range of phenotypic, metabolic, and molecular studies. Demographic data is collected for each subject, and all specimens stored in an NIH repository. A comprehensive literature review of the major molecular changes in preneoplastic breast tissue to define the carcinogenic pathway and aid in the molecular analysis of normal risk and high risk breast tissue has been conducted. Four types of preneoplastic breast tissue - normal/benign breast tissue at low risk for breast cancer, normal/benign tissue at high risk, epithelial hyperplasia, and epithelial hyperplasia with atypia were reviewed to define numerical chromosomal changes, structural chromosome changes, and changes in expression of individual genes. Patterns of molecular changes have been correlated with risk and with progression between histologic/morphologic subtypes. This review indicated that the earliest molecular changes in breast carcinogenesis are loss of heterozygosity and DNA methylation of tumor suppressor loci, present in morphologically normal low risk, and more frequently high risk breast tissue, while aneusomy is a later event and is first identified in high risk normal breast tissue. Gene amplification is an uncommon early event in preneoplastic tissues. This characterization provides an important standard for whole genome analysis of human breast tissue at risk for breast cancer. Proliferation of normal breast epithelial cells at increased risk for breast cancer is a critical determinant for clonal expansion and for the accumulation of genetic abnormalities in breast carcinogenesis. To understand regulation of this proliferation, the effects of two prominent stimulatory and risk factors for breast cancer, estradiol (E2) and insulin-like growth factor-1 (IGF-1) on normal and high risk breast epithelial cells was studied. IGF-1 stimulated growth of all breast epithelial cells in a time-dependent and dose-dependent manner without modulation of apoptosis. This proliferative action by IGF-1 was accompanied by the rapid stimulation of phosphorylation of IGF-1R and IRS-1, and by downregulation of IRS-1 at the posttranscriptional level (protein expression) and of IRS-2 at the transcriptional level (gene and protein expression). These cells express estrogen receptor alpha (ERalpha) and beta (ERbeta) and progesterone (PR) receptors, however estradiol did not stimulate proliferation or cell cycle progression and did not modulate ER or PR in any of these cells. Importantly, IGF-1 did not act synergistically with estradiol to stimulate any of these growth or metabolic processes, in contrast to the synergism observed in breast cancer. These findings indicate that IGF-1 is the dominant mitogen in early breast carcinogenesis. Estrogen responsiveness of normal breast epithelial cells and synergism with IGF-1 occur later in the carcinogenic pathway. The role of IGF-1 in enhancing chromosomal instability in early carcinogenesis will next be defined by studying its modulation of DNA damage repair and signaling in normal and increased risk breast epithelium.